Ca2+ ions serve as a signaling mechanism in almost all cell types to regulate numerous diverse functions. Ca2+ signals are ordered in a hierarchy, from openings of single Ca2+ channels ('fundamental' events), through the concerted openings of clustered channels ('elementary' events, such as Ca2+ puffs) to propagating Ca2+ waves, coordinated through Ca2+ diffusion and Ca2+-induced Ca2+ release. Fundamental and elementary events thus form the triggers and building blocks underlying the complex spatiotemporal Ca2+ signals that permit graded and selective regulation of cell functions. Our overall goals are to elucidate how the functional properties, spatial organization and interactions between Ca2-t- channels pattern spatiotemporal cellular signals. We focus on Ca2+ events underlying the inositol trisphosphate (IP3) signaling pathway, and Ca2+ flux through amyloid oligomer pores implicated in the pathophysiology of Alzheimer's disease. Capitalizing on recent advances in biophotonic technology, including total intemal reflection fluorescence and superresolution microscopy, we can now study these topics at the truly single- molecule level in intact cells. Our aims are to: (i) Further develop techniques for fast, 3-dimensional imaging Ca2+ flux through individual IPS receptor/channels (IP3Rs) in intact cells; for monitoring ER [Ca2+]; and for single-molecule localization of subtypes of native IP3Rs in transgenic mouse models, (ii) Elucidate the functional properties of IP3Rs in the intact cell, how the activity of channels is orchestrated to generate and temiinate elementary Ca2+ puffs, and to generate global Ca2+ waves, (iii) Resolve IP3R molecules with sub-micron precision to address hypotheses concerning the clustered organization and anchoring of these channels; their interactions via Ca2+ diffusion and allosteric mechanisms; and the putative function of 'silenf IP3Rs between puff sites, (iv) Investigate the molecular mechanisms by which amyloid oligomers form Ca2+- permeable pores by combining single-channel Ca2+ imaging with single-molecule photobleaching of fluorescent monomers to detennine pore stoichiometry; and elucidate the mechanisms by which intracellular oligomers induce Ca2+ liberation through IP3Rs. RELEVANCE (See instructions): Calcium sen/es a 'life or death' function in virtually all cells ofthe body, regulating processes as diverse as the heartbeat and synaptic transmission between brain cells, and is implicated in Alzheimer's and other diseases. Our goal is to elucidate the hierarchical mechanisms by which calcium signals are generated at the single-molecule level, with the dual aims of better understanding their normal functioning and how disruntions in r-alrJum signaling mav lead to disease.